| Objective:The subject mainly studies on quality analysis of Elaeagnus pungens leaf:the content determination of index components such as tiliroside,quercetin,kaemferol and the comparation of Elaeagnus pungens lesf from different areas to supply reference of the quality of Elaeagnus pungens leaf effectively,to comprehensively evaluate the quality of Elaeagnus pungens leaf by modern analytical instrument;to establish a best method for tiliroside about the extraction process; to estabilish a HPLC method and a UPLC method for simultaneous determination of quercetin,kaemferol in Elaeagnus pungens leaf from different areas and comparing the determination results. The study of fingerprint of Elaeagnus pungens leaf from different areas by HPLC and UPLC to distinguish the quality of Elaeagnus pungens leaf by the fingerprint; In Elaeagnus pungens leaf the determination of heavy metals and other microelements to reflect the quality of Elaeagnus pungens leaf; the study of common quality of Elaeagnus pungens leaf to comprehensive evaluate the quality of Elaeagnus pungens leaf.Methods: The content of index components such as tiliroside,quercetin,kaemferol in12 batches of Elaeagnus pungens leaf from different areas were respectively determined by HPLC, and established their quantitative methods; to establish a best method for tiliroside about the extraction process; the HPLC was used to establish the fingerprint of 12 batches of samples and analyzed by cluster analysis and similarity evaluation; this section mainly determined heavy metals(Pb、Cd、As、Hg、Cu) and other microelements(Fe、Mn、Cr、Mg、K、Zn) in 12 batches of Elaeagnus pungens leaf from different areas by AAS; study the common quality of Elaeagnus pungens leaf which including content determination of water, lixivium, general ash and indiscerptible ash in acid. Then the objective and sufficient quality evaluation methods were obtained.Results: The extraction process was optimized by single factor and orthogonal experiments, the best extraction processing mothed of tiliroside were as follows: the samples was extracted by 80% methanol(50m L), the backflow extraction time is 1.5h, the temperature is 85℃, extracting two times. HPLC condition of tiliroside: the column was Agilent C18(4.6mm×250mm,5μm), the mobile phase is acetonitrile(B)-0.1% phosphoric acid(A) at the flow rate of 1.0 m L·min-1, gradient elution process as follows: 0 ~ 5min,10% ~ 15%B;5 ~ 10 min,15% ~ 20%B;10 ~15min,20% ~ 25%B;15 ~ 20 min,25% ~ 28%B;20 ~ 25 min,28% ~ 28%B;25 ~30min,28% ~ 30%B;30 ~ 35 min,30% ~ 40%B; The column temperature was 30 ℃.The detection wavelength was set at 315 nm, The injection volume was 20μL. The linear range of tiliroside was 0.005~0.10 mg·m L-1(r=0.9999) with an average of(RSD=99.14%,n=6). Though orthogonal experiments determined the best process: the samples was extracted by 25% hydrochloric acid-ethanol(1:4)(100m L), the backflow extraction time is 1.5h, the temperature is 85℃, extracting once. HPLC condition of quercetin and kaemferol: the column was Agilent C18(4.6mm×250mm,5μm), the mobile phase consisted of acetonitrile(B)-0.1% phosphoric acid(A) at a flow rate of 1.0 m L·min-1, gradient elution process as follows: 0 ~ 5min,10% ~ 15%B;5 ~ 10 min,15% ~ 20%B;10 ~ 15 min,20% ~ 25%B;15 ~20min,25% ~ 28%B;20 ~ 25 min,28% ~ 28%B;25 ~ 30 min,28% ~ 30%B;30 ~35min,30%~40%B; The column temperature was 30 ℃.The detection wavelength was set at 366 nm, The injection volume was 10μL.with this HPLC condition to detect the reference substance solvent and test samples solvent, it seems that the quercetin and kaemferol in the reference substance solvent and test samples solvent have a good separating degree with the other chromatographic pesk, The linear range of quercetin and kaempferol were 2.2~44μg·m L-1(r=0.9999) and 3.92~98μg·m L-1(r=0.9999)with an average of(RSD=1.69%,n=6) and(RSD=1.11%,n=6).The contemt of quercetin and kaempferol from different habitata ranged from 0.3795~2.4628mg·g-1 and 2.8643~8.0797 mg·g-1. The HPLC fingerprint chromatographic profile of Elaeagnus pungens leaf were from different areas. The data from HPLC was analyzed by similarity evaluation and system cluster analysis. Get the peaks of common peaks HPLC fingerprint, get the peaks of common peaks UPLC fingerprint, and making sure the rutin,kaemferol,tiliroside peak. The similarity of HPLC fingerprint is to 0.8,. The establishment of the methods fingerprints of Elaeagnus pungens leaf to evaluate the quality. This section mainly determined heavy metals and other microelements in 12 batches of samples by AAS. The samples from all the areas have rich microelements, but have differences in 12 batches of samples. According to “the import and export of medicinal plants and preparation of green industry standard”, the test results showed that the content of heavy metals of all the 12 samples were not exceed the limitation. According to 2010 Chinese Pharmacopoeia, the common quality of Elaeagnus pungens leaf was studied which including content determination of water,lixivium,general ash and indiscerptible ash in acid. All the samples conformed to prescribed,the content of lixivium were between4.10%-7.15%.Conclusion: Over the systematic research of Elaeagnus pungens leaf, the quality control methods by several indexes are established. And the HPLC and UPLC fingerprint quality evaluation methods are established. Determined the heavymetal and other microelements contents and the common quality of Elaeagnus pungens leaf was studied. The quality of Elaeagnus pungens leaf was comprehensive evaluated. and can overall control the quality of Elaeagnus pungens leaf. |
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